A major function of the enzyme intestinal alkaline phosphatase (iAP) is the detoxification of lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4). Hence, iAP has a role in the defence of maintaining intestinal barrier integrity. As intestinal barrier integrity is impaired in coeliac disease (CD), we tested the expression and localization of iAP in duodenal mucosa specimens from children with newly diagnosed CD (n = 10), with CD on gluten-free diet (GFD) (n = 5) and compared to those from ten healthy children. The mRNA and protein expression was determined by RT-PCR and Western blot analysis, respectively. Tissue localization of iAP and TLR4 was determined by immunofluorescence staining. iAP protein expression level was significantly lower than normal in newly diagnosed CD, while it was normalised in children on GFD. iAP and TLR4 colocalized at the epithelial surface of duodenal mucosa in each group of subjects enrolled. The finding of decreased iAP protein levels in newly diagnosed CD is consistent with its role in decreased intestinal barrier integrity. The latter may be the result of decreased LPS-detoxifying ability.
Coeliac disease (CD) is a chronic enteropathy that is triggered by chronic exposure to gluten protein of wheat, barley and rye. Both the innate and the adaptive parts of the immune system are involved in its pathogenesis in genetically predisposed individuals [1, 2]. The diagnosis of CD is based on serologic tests, mucosal biopsy and the effects of gluten-free diet (GFD) on the symptoms . The innate immune system in the pathogenesis of CD includes Toll-like receptors (TLR), a family of bacterial and viral pattern recognition receptors . Under physiological circumstances TLRs play a crucial role in preserving a regulated homeostasis within the gut by the elimination of pathogens and toleration against commensal microbes . TLR4, a member of the transmembrane receptor TLR family, is activated by lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria . The uncontrolled activation of TLR4 may induce mucosal loss and induction of a proinflammatory cascade . Recently, our group has shown increased mRNA and protein levels of TLR4 in the duodenal mucosa of children with newly diagnosed CD, and these levels further increased in CD patients on GFD . In addition, we found a higher prevalence of TLR4-positive dendritic cells and monocytes in the peripheral blood of patients newly diagnosed with CD compared to controls .
The enzyme intestinal alkaline phosphatase (iAP) is expressed in large quantity on the apical surface of the enterocytes . iAP may play a pivotal role in the maintenance of intestinal barrier integrity by detoxification of LPS, the ligand of TLR4 [11, 12]. iAP is able to dephosphorylate the disphosphoryl lipid A part of LPS. This avoids pathological activation of TLR4 and the MyD88-dependent signalling events . The precise role of iAP in CD is not defined, but according to previous studies in an animal model of inflammatory bowel disease, exogenously administered iAP improved the histological signs of inflammation . A recent clinical study also reinforced the beneficial effects of exogenous iAP products in inflammatory bowel disease patients . For CD, the data available for iAP suggest that iAP enzyme activity decreases in the duodenal biopsy specimens and strongly correlates with histological signs , but the iAP protein levels were not investigated.
The aim of our study was to collect additional data on iAP protein and mRNA expression in CD. We also tested whether or not iAP and TLR4 are colocalized in duodenal mucosa.
Materials and methods
Patients and duodenal biopsies
We enrolled ten children (two boys, eight girls; median age, 4 years; range, 2–12 years) with newly diagnosed CD and five children (two boys, three girls; median age, 12 years; range, 6–13 years) with GFD-treated CD and ten controls (five boys, five girls; median age, 9.5 years; range, 2–16 years) in the study. CD was diagnosed by the criteria of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition [17, 18]. Screening for CD was performed using IgA anti-tissue transglutaminase . High level of anti-tissue transglutaminase and villous atrophy was demonstrated in all patients with newly diagnosed CD. Children on GFD had full clinical and histological remission supported by normal blood levels of anti-tissue transglutaminases. The time frame between the first and the second biopsies of patients on GFD (GFD-treated CD) was 1.5 years (range, 0.5–2.5 years). The control group included children with chronic abdominal pain or chronic diarrhoea referred to the outpatient clinic. Normal blood levels of anti-tissue transglutaminases were demonstrated, and no histological alterations were present in their duodenal biopsy specimens. The subjects' anthropometric parameters in each group were comparable (Table 1). Written informed consent was obtained from parents prior to the procedure. The study was approved by the Semmelweis University Regional and Institutional Committee and Research Ethics.
RNA isolation and real-time PCR
Total RNA was isolated from the duodenal biopsy samples. Total RNA was reverse-transcribed to generate first-strand cDNA. iAP mRNA expression was determined by real-time PCR on a Light Cycler480 (Roche Diagnostics, Mannheim, Germany). PCRs were performed containing RealTime ready Catalog Assay primer (Roche Diagnostics), Brilliant II QPCR Master Mix (Stratagene, Cedar Creek, TX, USA) and cDNA. Conditions for iAP include one cycle, 95°C, 10 min (denaturation), followed by several cycles at 95°C, 20 s and 60°C, 1 min (annealing and extension). The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mRNA expression of GAPDH was determined using Brilliant II Fast SYBR Green QPCR Master Mix (Stratagene), PCR primers (forward: 5-CAC CAC CAT GGA GAA GGC TG-3′; reverse: 5-GTG ATG GCA TGG ACT GTG-3′, Invitrogen, CA, USA) and cDNA. Conditions for GAPDH include one cycle, 95°C, 2 min followed by 50 cycles at 95°C, 20 s, and 60°C, 40 s. Results were analysed by Light-Cycler software 480 (Roche Diagnostics).
Protein isolation and Western blotting
Duodenal biopsy samples were homogenized in lysing solution, and the total protein concentrations were analysed by DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). An equal amount of protein samples, 0.5 μg from each sample, was separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (120 V, 40 mA, 120 min) (Penguin™ Dual-Gel Water Cooler Systems, Owl, NH, USA) and transferred to nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) (70 V, 220 mA, 120 min) (MiniTank™ electroblotter, Owl). Membranes were blocked in 1% fat-free dry milk solution (1 h) and incubated with iAP-specific rabbit polyclonal antibody (1:1,000, 1 h) (AbCam, Cambridge, UK). Equal protein loading was confirmed by ß-actin-specific (C-11) goat polyclonal IgG antibody (1:100) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The membrane was incubated with peroxidase-conjugated secondary anti-rabbit IgG or donkey anti-goat IgG antibodies (1:2,000, 30 min) (Santa Cruz Biotechnology Inc.). Immunoreactive bands were visualized using the enhanced chemiluminescence Western blotting detection reagents (GE Healthcare). Bands were analysed with software Image J. 1.42q (National Institutes of Health, USA). The values were expressed as relative optical density.
The snap-frozen duodenal biopsy samples were imbedded in Shandon cryomatrix (ThermoElectron Co., Waltham, MA, USA) and cut to 3–4-μm slides. For iAP and TLR4 double immunofluorescence, iAP-specific rabbit polyclonal antibody and TLR4-specific goat polyclonal antibody were applied (1:100, 1 h) (Abcam Plc). Alexa Fluor 488 donkey anti-goat and Alexa Fluor 568 goat anti-rabbit antibodies were used as secondary antibodies (Invitrogen). Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss, Jena, Germany) was used with ×20 Plan Apochromat (NA = 0.8) and ×63 Plan Apochromat oil immersion differential interference contrast objectives (NA = 1.4).
Data were analysed using Statistica 7.0 software (StatSoft Inc., Tulsa, OK, USA). After testing the normality with Shapiro–Wilk's test, nonparametric Mann–Whitney U test was used. Values of p ≤ 0.05 were considered to be significant and expressed as mean±SD.
iAP mRNA expression
Real-time PCR was performed to determine the mRNA expression of iAP in children with newly diagnosed CD, children with GFD, and healthy controls. As shown in Fig. 1, iAP mRNA expression was decreased in the duodenal mucosa of children with newly diagnosed CD compared to healthy controls; however, the difference failed to reach significance. There was no significant difference in children maintained on GFD and newly diagnosed CD groups, but the iAP mRNA expression of the GFD group has started to normalise.
iAP protein levels
We performed Western blot analysis in the duodenal biopsy specimens from newly diagnosed CD patients, children with GFD, and healthy controls, which revealed one distinct band at 60 kDa. The amount of iAP protein in newly diagnosed children with CD was significantly decreased when compared to controls (33%, p < 0.05) (Fig. 2). iAP protein levels of patients on GFD were higher than those of CD patients but lower than those of controls (Fig. 2).
Localization of iAP and TLR4 in the duodenum
Immunofluorescence staining was performed to investigate the possible colocalization of iAP and TLR4 in patients with CD, children with GFD, and healthy controls (Fig. 3). iAP and TLR molecules clearly colocalized in a dot-like pattern in all groups studied. iAP was restricted to the epithelial surface of the duodenal mucosa, but no immunofluorescent signal was detected in goblet cells, in immune cells of the lamina propria or in the Lieberkühn cells.
To the best of our knowledge, we are the first to demonstrate the alteration of protein expression of iAP and the localization of iAP and TLR4 in the duodenal mucosa of children with newly diagnosed CD, children maintained on GFD, and healthy controls. We found significantly reduced iAP protein levels in the duodenal mucosa of newly diagnosed CD patients, compared to healthy controls. The iAP protein levels of children maintained on GFD were lower than those of controls, but higher than those of patients with CD.
iAP is a recently identified enzyme with a major role in intestinal barrier integrity. Its possible contribution to CD is highlighted by Prasad et al. who reported that the activity of iAP and other brush-border enzymes correlates with histologically moderate and severe lesions in CD duodenal specimens  and that these abnormalities vanish on GFD [20, 21]. In line with these results, we found decreased iAP protein levels in newly diagnosed patients with CD that tended to normalise on GFD. We have also shown in a previous study that the increased expression of tight-junction molecules such as claudin 2 and 3 might contribute to the change of barrier function of the duodenal mucosa  and to increased permeability and consequential malabsorption. The decreased level of iAP may be a further element contributing to the degradation of intestinal barrier integrity . Nevertheless, the decreased iAP level in untreated patients with CD is probably an inflammation-related, nonspecific finding for CD; however, our results (colocalization with TLR, TLR–LPS-detoxifying ability) may add new pieces to the puzzle of the pathomechanism of CD.
The possible contribution of iAP to intestinal barrier integrity is thought to be mediated by its action on TLR ligands. Indeed, we observed that iAP and TLR4 occur in a colocalized manner. Previously, we have shown increased TLR4 expression in newly diagnosed patients with CD . Theoretically, the lower-than-normal iAP together with a higher-than-normal TLR4 expression results in an imbalance in iAP/TLR4 ratio and, hence, an increased inflammatory response to LPS present in the gut. These data support that innate immunity and, particularly, TLRs are major players in disturbed intestinal barrier integrity in CD.
In summary, we found decreased iAP duodenal mucosal protein expression in children with newly diagnosed CD. In addition, a strong colocalization of iAP and TLR4 at the epithelial surface of duodenal mucosa was found. These findings suggest that iAP plays an important role in the restitution of mucosal barrier integrity and in innate immunity.
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We thank Mária Bernáth for her excellent technical assistance. This work was supported by grants OTKA-76316, OTKA-K81117, TÁMOP 4.2.2. B-10/1-2010-2013, and ETT-028-02. Gábor Veres and Ádam Vannay are holders of the János Bolyai Research grant; this article was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.
Conflict of interest
We declare that we have no conflict of interest.
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Molnár, K., Vannay, Á., Sziksz, E. et al. Decreased mucosal expression of intestinal alkaline phosphatase in children with coeliac disease. Virchows Arch 460, 157–161 (2012). https://doi.org/10.1007/s00428-011-1188-5
- Intestinal alkaline phosphatase
- Toll-like receptor
- Duodenal biopsy
- Coeliac disease